PNH Clone Research Articles

RESULTS OF LONG-TERM THERAPY WITH A BIOSIMILAR OF ECULIZUMAB IN PATIENTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA.

The study aimed to assess the safety, immunogenicity, and efficacy of long-term therapy with biosimilar of eculizumab (Elizaria®) in PNH patients. The study included 30 patients with PNH who had completed previous clinical trials. Of these, 25 patients continued receiving the biosimilar product, and 5 patients switched from the originator product Soliris. The maximum duration of follow-up was 104 weeks, during which the investigational product was administered 52 times at a standard dose. Throughout the study, the levels of LDH, hemoglobin, reticulocytes, and PNH clone remained stable compared to baseline, regardless of the previous therapy (p > 0.05). There were no significant differences in the number of patients with chronic kidney disease at different visits, as well as in the number of patients who received donor red blood cell and platelet transfusions during the study (p > 0.05). There were two cases of adverse reactions reported in two patients (6.6%): elevated aspartate aminotransferase (3.3%) and alopecia (3.3%). Immunogenicity analysis showed no significant differences in the frequency of anti-drug antibody detection compared to baseline (p > 0.05). The study findings confirm the long-term efficacy and safety of biosimilar in patients with PNH.

Genetic Variation of SH2B3 in Patients with Myeloid Neoplasms

To observe the genetic variation of SH2B3 in patients with myeloid neoplasms. The results of targeted DNA sequencing associated with myeloid neoplasms in the Department of Hematology, Xuanwu Hospital, Capital Medical University from November 2017 to November 2022 were retrospectively analyzed, and the patients with SH2B3 gene mutations were identified. The demographic and clinical data of these patients were collected, and characteristics of SH2B3 gene mutation, co-mutated genes and their correlations with diseases were analyzed. The sequencing results were obtained from 1 005 patients, in which 19 patients were detected with SH2B3 gene mutation, including 18 missense mutations (94.74%), 1 nonsense mutation (5.26%), and 10 patients with co-mutated genes (52.63%). Variant allele frequency (VAF) ranged from 0.03 to 0.66. The highest frequency mutation was p.Ile568Thr (5/19, 26.32%), with an average VAF of 0.49, involving 1 case of MDS/MPN-RS (with SF3B1 mutation), 1 case of MDS-U (with SF3B1 mutation), 1 case of aplastic anemia with PNH clone (with PIGA and KMT2A mutations), 2 cases of MDS-MLD (1 case with SETBP1 mutation). The other mutations included p.Ala567Thr in 2 cases (10.53%), p.Arg566Trp, p.Glu533Lys, p.Met437Arg, p.Arg425Cys, p.Glu314Lys, p.Arg308*, p.Gln294Glu, p.Arg282Gln, p.Arg175Gln, p.Gly86Cys, p.His55Asn and p.Gln54Pro in 1 case each. A wide distribution of genetic mutation sites and low recurrence of SH2B3 is observed in myeloid neoplasms, among of them, p.Ile568Thr mutation is detected with a higher incidence and often coexists with characteristic mutations of other diseases.

Diagnostic landscape of first-time cytometric screening for paroxysmal nocturnal hemoglobinuria in Poland in 2013–2022

BackgroundParoxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disorder characterized by PIG-A mutations, leading to glycophosphatidylinositol (GPI)-anchored proteins deficiency that triggers hemolysis – a hallmark of the disease. PNH diagnostics is based on high-sensitivity multicolor flow cytometry (MFC), enabling to detect even small populations of PNH cells. In this single-center, retrospective study, we aimed to characterize a cohort of PNH clone-positive patients first time screened from January 1st, 2013 until December 31st, 2022 with MFC according to International Clinical Cytometry Society PNH Consensus Guidelines.ResultsOut of 2790 first-time screened individuals, the presence of PNH clone in neutrophils was detected in 322 patients, including 49 children and 273 adults. Annual incidence was stable at a median of 31 patients (14 and 19 with clone sizes ≤ 1% and > 1%, respectively), with a decline in number of patients with clone sizes > 1% observed in 2020, potentially influenced by the COVID-19 pandemic. The most common screening indications were aplastic anemia and other cytopenias.ConclusionsA significant underrepresentation of hemolytic patients was observed as compared to the published cohorts suggesting that these patients are missed in diagnostic process and classic PNH remains underdiagnosed in Poland.

Long-Term Outcomes of Immunosuppressive Therapy for Aplastic Anemia: A Single-Center Experience

Background. Bone marrow transplantation-ineligible aplastic anemia (AA) is most effectively treated with combined immunosuppressive therapy (IST). It yields remissions in most patients. However, it has such disadvantages as frequent relapses, incomplete hematologic recovery, and clonal evolution risk. Besides, АА is not always treated according to standard regimens. For different reasons, some AA patients receive delayed therapy or IST mono-treatment predominantly with cyclosporine A (CsA).
 Aim. To assess long-term IST outcomes in AA patients followed-up at the Russian Research Institute of Hematology and Transfusiology for 5 years after therapy onset.
 Materials & Methods. The study enrolled 30 AA patients who received IST for more than 5 years (continuous follow-up of 5.5–33 years) with monitoring of the main hemogram parameters and PNH clone size. Patients were aged 19–73 years (median 29 years). There were 8 women and 12 men. Based on international criteria, severe AA (SAA) was initially diagnosed in 18 patients, and non-severe АА (NAA) was diagnosed in 12 patients. Combined IST was administered to 22 patients (18 SAA patients and 4 NAA patients), the remaining 8 patients received ATG (n = 1) and CsA (n = 7).
 Results. A response to IST was achieved in 28 (93.3 %) out of 30 patients, 16 (53.3 %) of them showed complete remission. This paper documents the characteristics of hematologic recovery depending on the compliance with standard therapy regimens, as well as on the disease variant, development of late complications and clonal evolution, characteristics of pregnancy and childbirth in 4 female patients in remission. PNH clone increased in more than a half (10 out of 16) patients whose clone was initially > 2.6 %. Long-term clonal evolution to myeloid neoplasia (13 years after IST onset) was registered in 2 (6.7 %) patients with complete AA remission. Aseptic (avascular) osteonecrosis as complication was reported in 6 (20 %) followed-up patients.
 Conclusion. The results of the study highlight the importance of and the need for early start and adherence to standard combined IST regimens aimed at optimum therapeutic effect in both SAA and NAA patients, as well as for long-term follow-up of patients after completing IST.

Prevalence of Paroxysmal Nocturnal Haemoglobinuria Clone in Aplastic Anaemia: A Single Centre Study

Background: Flow cytometry assay for PNH clone is a compulsory routine test for all aplastic anaemia patients. Objective: To estimate the frequency of PNH clone in aplastic anaemia. Method: Twenty-two known cases of aplastic anaemia patients were enrolled for the study. Flow cytometric quantitation of glycosyl phosphatidyl-inositol (GPI)-anchored proteins deficiency using markers CD14, CD24, CD45, CD59, Fluorescent Aerolysin (FLAER), CD235a (6 markers) were performed. Result: PNH clone was identified in 8 (36.4%) of the study population. Among PNH clone positive patients 7 (87.5%) were suffering from non-severe aplastic anaemia. Conclusion: PNH clone is significantly associated with aplastic anaemia and PNH clone assay should be regularly assayed in aplastic anaemia patients for specific management.

Diagnosis and treatment of aplastic anemia and lower-risk myelodysplastic neoplasms

Myelodysplastic neoplasms (MDS) are clonal hematological malignancies arising from gene mutations. Immunosuppressive therapies (IST) are effective in lower-risk MDS (LR-MDS) with characteristics such as hypoplastic marrow with low blasts or low ring sideroblasts, and with a small increase of PNH clones or decrease of megakaryocytes. Differential diagnosis of these LR-MDS cases from AA can be difficult, and precise diagnosis requires careful evaluation of bone marrow cellularity and dysplasia. To decide on an appropriate treatment strategy for LR-MDS, it is important to evaluate the underlying pathology, and preferentially select IST as first-line therapy in patients with features that indicate immune-mediated bone marrow failure.

A rare case of compound Hemoglobin Lansing/Hemoglobin S coinherited with alpha thalassemia trait (-alpha3.7) presented with spurious hypoxemia and moderate hemolytic anemia

Abstract We present a case of a 27-year-old female at 27 weeks of gestation, who presented at the emergency department for vaginal spotting. She was also found to have low oxygenation on pulse oximetry of approximately 82% at room air. Her SpO2 did not improve with O2 supplement. She had a similar presentation a month before, with mild light-headedness and low oxygenation. However, two repeated air blood gas (ABG) findings were normal for oxygen saturation. Hemoglobinopathy was suspected due to the disparity. She was anemic during her presentation, with hemoglobin 8.3 g/dl, RBC count 3.36 million/ul, MCV 81 fl and MCH 24.7 pg. Additional workup showed increased reticulocyte count, decreased haptoglobin, negative direct Coombs test, negative G6PD test, and no evidence of a PNH clone. Capillary hemoglobin electrophoresis showed HbA 59.8%, HbS 34.5%, HbA2 3.6% and a small peak (2.1%) in the Z8 zone with a trailing edge at HbF window. HPLC revealed an “unknown peak” of 8.2% with a retention time of 1.93 minutes as well as the HbS peak. There was also a tiny additional A2 peak unresolved from the main A2 peak. Alpha globin gene sequencing revealed one copy of the -alpha3.7 alpha-globin deletion and one copy of Hb Lansing variant in the alpha2-globin gene. This is consistent with the compound Hemoglobin Lansing/Hemoglobin S coinherited with alpha thalassemia trait. Hemoglobin Lansing is a rare unstable alpha hemoglobin variant with mutation of c.264C>G resulting with a substitution of His by Gln at codon 87. The mutation was first discovered in the alpha 2 gene. Then the same mutation was found in alpha 1 gene later and named as Hemoglobin Lansing-Ramathibodi. To the best of our knowledge, this is the first reported case of Hemoglobin Lansing with alpha thalassemia trait (-alpha3.7) and HbS. Spuriously low pulse oximetry measurements have previously been reported in Hb Lansing variants. It was speculated to be due to different absorption spectra of Hb Lansing which is under study now. The patient also presented with hemolytic anemia. The exact cause was undetermined yet although it might be related to her hemoglobinopathy. Mild hemolytic anemia was reported in two cases of co-inheritance of Lansing-Ramathibodi and Hb Pakse (a non-deletional alpha thalassemia) but not in the members of the same family with Hb Lansing alone. It was also reported that some unstable alpha chain variants, when associated with another alpha thalassemia defect, present with a more severe phenotype including hemolytic anemia even though essentially asymptomatic in a heterozygous state.

Neutrophil PNH Type II Cells Are Associated with Thrombosis and Bone Marrow Failure without Hemolysis: Evidence from Analysis of the 5-Year French Nation-Wide Multicenter Observational Study

Introduction Flow cytometry (FCM) is the gold standard test to diagnose a PNH clone, type II and III cells being defined by a partial or a complete deficiency, respectively, in GPI-linked proteins. The relevance of classifying type II and type III PNH cells on WBC, unlike their counterparts among red blood cells, remains unknown. Nevertheless, exploring this distinction could hold potential significance for clinical decision-making and patient management. We aimed to assess the distribution of type II and type III cells on WBC in patients displaying a major PNH clone (defined as GPI-deficient neutrophils above 50%) and to investigate their association to clinical presentations. Methods The 5-year French nation-wide multicenter observatory (2016-2021) gathered all patients with a diagnosed PNH clone or GPI-deficient cells ≥0.01% on neutrophils, detected in France by FCM. Only patients with a major PNH clone >50% were included in this study. For each patient, the baseline assessment was considered as the initial PNH clone detection, which facilitated long-term follow-up. A referent cytometrist at each center provided clinical and biological information as well as FCM raw data files. This study was approved by the national research ethics board. Fisher, chi-square and ANOVA tests were performed to compare the median values and frequencies. Results As of February 28 th, 2021, 41 participating FCM laboratories across France had enrolled 637 patients with a PNH clone or GPI-deficient cells ≥ 0.01%. Among 109 patients displaying a major PNH clone (i.e., ≥ 50%) on neutrophils, 100 patients with distribution of type II and type III cells on neutrophils were validated by the two principal investigators(Figure 1). We performed an AUROC test based on the presence or absence of hemolysis and determined a 3.9% threshold of type II neutrophils. This enabled the distinction of two groups at baseline: 14 patients with high type II cells (7.76%; IQR, 5.63-13.63) and 86 patients with low type II cells (0.05%; IQR, 0.00-0.71; p<0.0001). Besides, type II monocytes were higher in the “high type II” group (6.62%; IQR, 5.30-22.45) compared to the “low type II” group (0.00%; IQR, 0.00-0.71; p<0.0001). However, the total clone size was similar for both groups on WBC (median size on neutrophils: 91.51% (IQR, 80.47-96.90) vs 90.85% (IQR, 79.55-98.20); p=0.79, on monocytes: 89.88% (IQR, 80.27-92.24) vs 91.24% (IQR, 80.59-96.73); p=0.98). The patient median age at diagnosis was 51 years [24-79] for “high type II” group and 40 years [18-85] for “low type II” group and a M/F sex ratio of 1.0 vs 1.3. Diagnoses were confirmed between 2004 and 2021 with clinical information available in 96% of cases. Hemolysis was more frequent in “low type II” group: 96.0% (72/75) compared to “high type II” group: 50.0% (7/14) (p<0.0001) for the 89 patients with available data on hemolysis status, with a trend to more elevated LDH (1289 UI/L; IQR, 553-1931 vs 735 UI/L, IQR: 459-1037; p=0,052) and a reduced clone size on red blood cells (3.26%; IQR, 1.20-32.46 vs 30.48%; IQR, 16.47-45.02; p=0.097). Interestingly, thrombosis appears to be more frequent in “high type II” group: 35.7% (5/14) vs 7.3% (6/82) in the “low type II” group (p<0.01). Bone marrow failure (BMF) without hemolysis (aplastic anemia or MDS) appears to be also more frequent in “high type II” group: 42.9% (6/14) vs 2.7% (=2/75) in the “low type II group” (p=0.0001). All complete blood count parameters, including hemoglobin levels, were not significantly different between the 2 groups. Among the 62 out of 100 patients who displayed at least one follow-up point, 57 have been treated with anti-C5. Long-term follow-up up to 10.5 years showed that 22 patients displayed an increase in the total clone size (mean: +18.3%, Figure 2), 27 remained stable (+0.3%) and 8 decreased (-29.6%). Interestingly, we did not observe any major modification of type II neutrophil percentages regardless of the total clone size evolution (+0.2%; -0.1%, +0.2%). Conclusion This multicenter study based on robust FCM analysis showed for the first time the value of accurate quantification of neutrophil PNH type II cells as patients with high type II cells above 3.9% displayed less hemolysis, more thrombosis and more BMF without hemolysis, which suggests a closer follow-up may be warranted for these patients.

Single-Cell Dissection Reveals a Distinct Origin of Small Paroxysmal Nocturnal Hemoglobinuria Clones in Immune Aplastic Anemia and Healthy Individuals

Abstract Introduction Paroxysmal nocturnal hemoglobinuria (PNH) clones are characterized by the absence of cell surface glycosylphosphatidylinositol (GPI)-anchors owing to somatic mutations in the PIGA gene on the X chromosome. Large PNH clones derived from PIGA mutant multipotent progenitors (MPPs) result in clinically significant hemolysis. In immune aplastic anemia (AA), the PNH clone represents immune escape from T cell-mediated pathogenesis and is usually small (<1%), sometimes likely derived from lineage-committed progenitors (LCPs), with detectable GPI-anchor deficient (PNH-type) cell populations only in granulocytes and monocytes. Small PNH-type cells are also present in healthy individuals at a much lower frequency (<0.003%) than in AA patients. The nature of the small PNH-type cells is not fully understood because of the difficulties in analyzing rare cell populations. Methods In this study, we developed a method to detect PIGA mutations within a small population of cells at single-cell resolution. PNH-type cells were enriched from peripheral blood leukocytes using magnetic microbeads and monoclonal antibodies specific for GPI-anchored proteins. The enriched cell fraction was then subjected to fluorescence-activated cell sorting after staining with lineage-specific markers and fluorescein-labelled proaerolysin. We sorted a limited number (100 cells in males and 50 cells in females) of PNH-type granulocytes (PNH-Gs) and T cells (PNH-Ts) as well as wild-type cells into separate tubes, and their PIGA genes were directly amplified by multiplex PCR without a DNA extraction procedure for subsequent deep nucleotide sequencing. To ensure the specificity of PIGA mutation detection, we set cut-off thresholds of 2% for single-nucleotide substitutions and 1% for indels, in addition to the thresholds based on base-position-specific error rates in 10 sets of 2000 wild-type cells, as previously reported (Zaimoku et al. Blood 2021). Results In 19 sets of 50-100 wild-type cells from 13 individuals, we detected only 7 mutations with variant allele frequencies (VAFs) ranging from 2.1% to 4.2% (Figure 1). Three of these were synonymous mutations, and the remaining 4 were missense mutations, suggesting that these mutations may be true passenger mutations rather than sequencing errors. In all 6 healthy individuals, small PNH-type cells exhibited a polyclonal pattern. We detected numerous small, likely inactivating, PIGA mutations in PNH-Gs, with a median of 11 (range, 6-18) mutations per 100 cells. In 3 individuals assessed twice, approximately half of the PIGA mutations were detected in both sets of PNH-Gs, whereas the remaining mutations were unique to each set. Similarly, PNH-Ts from 4 healthy individuals showed polyclonality, with a median of 14 (range, 6-22) mutations per 100 cells. Interestingly, PIGA mutations were not shared between PNH-Gs and PNH-Ts, except for 2 splice site mutations with VAFs <10%. The polyclonal pattern was further confirmed by Sanger sequencing, which successfully detected 7 unique PIGA mutations in 5 sets of 5 PNH-type cells (instead of 100 cells) in a healthy donor, whereas no mutations were detected in 3 sets of 5 wild-type cells, except for 1 synonymous mutation. In contrast, small PNH-Gs of 5 AA patients, with frequencies of 0.02%, 0.08%, 0.09%, 0.2%, and 0.5% of total granulocytes, were primarily derived from single PIGA mutant clones, which accounted for 90%-99% of PNH-G populations. In another patient with AA, PNH-Gs with a frequency of 3.3% were derived from 2 dominant clones. Notably, the dominant PIGA mutations in the PNH-Gs of AA patients were all detectable in PNH-Ts, even in 2 patients whose PNH-Ts were not detectable (<0.003%) and in 3 patients whose percentages of PNH-Ts were 0.004%, 0.02%, and 0.08% before microbead-based enrichment. Discussion Our novel sequencing approach provides insight into the clonality and lineage distribution of small PNH-type cells without in vitro culture manipulation. In healthy individuals, most PNH clones originate from LCPs, where many cell divisions occur, thereby providing opportunities for the acquisition of PIGA mutations. In immune AA, only PIGA mutations acquired at the MPP level lead to clonal proliferation, strongly suggesting that the immune pathogenesis of AA targets MPPs, rather than LCPs. A clonality analysis of small PNH-type cells thus helps diagnose the immune pathogenesis of AA.

Predicting Clonal Evolution to Secondary Myeloid Neoplasms in Aplastic Anemia through Machine Learning

Background: One of the major long-term complications in aplastic anemia (AA) is the clonal evolution to secondary myeloid neoplasms (sMNs) such as acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) 1. Hematopoietic stem cell transplantation (HSCT) is largely considered a curative treatment option for AA and is effective at preventing the clonal evolution of sMNs 2. However, patients over 40 years old or those with poor donor options are contraindicated for HSCT due to elevated post-transplant mortality rates 3. When treated with non-transplant strategies, patients with AA have indeed a high risk for acquisition/expansion of somatic myeloid mutations, with some harbingers of future clonal progression to full-blown MDS or AML. Older age, presence of myeloid mutations other than BCOR/L and PIGA at onset, and lack of response to standard immunosuppressive treatment (IST) have been identified as factors predictive for subsequent sMN evolution 4,5,6. If an individual patient's risk of developing a sMN could be assessed through a predictive model that uses information collected at initial AA diagnosis, providers may consider HSCT for traditionally contraindicated patients. However, no such predictive model that could aid in risk assessment currently exists. Methods: In our study, we utilized a large, multi-institutional cohort of patients diagnosed with AA to generate a comprehensive dataset. This dataset contained 76 variables, encompassing relevant clinical and molecular annotations. On this dataset, we trained a machine learning (ML) model to predict patients who would evolve sMNs. Variables with the highest feature impact score (FIS) in predicting clonal evolution to sMN were then chosen to be included in the final ML model, and the algorithm's fitness was calculated using the Leave-One-Out cross-validation method. Variables missing in more than 30% of subjects were imputed using K-Nearest Neighbors (N=20). Of our initial cohort of 455 patients, 213 patients were excluded from the study. 35 patients were excluded because they were treated with HSCT. 39 pediatric cases (defined as <18 years old at the time of diagnosis) were excluded from the study. 91 patients were removed for not receiving specific treatment (IST or HSCT). The remaining 77 patients were excluded due to having a sMN detected at the time of or prior to AA diagnosis, insufficient data, or detection of a germline mutation indicative of an inherited pathophysiology. Our final cohort included a total of 242 acquired AA patients from UT Southwestern Medical Center and Cleveland Clinic. Results: Among our final cohort of 242 patients, 119 (49.2%) were male. The cohort's mean and median ages at AA diagnosis were 53.1 years (SD = 18.9 years) and 58.1 years, respectively. The median follow-up time was 4.04 years. 80 (33.1%) of our patients had a clinically significant PNH clone (defined as >0.001%). 29 patients developed sMNs, with 24 developing MDS and 5 developing AML. Of patients who underwent clonal evolution to sMNs, the median age at the time of AA diagnosis and sMN diagnosis was 63.1 years and 67.2 years, respectively. The median time elapsed from initial AA diagnosis to sMN evolution was 3.78 years. The median time elapsed between initial AA diagnosis and earliest IST administration was 51 days. Chi-squared analysis showed that patients who received ATG treatment were more likely to develop sMNs, with 78.0% of all patients receiving ATG, while 96.6% of patients who developed sMNs had received ATG (p=0.00927). Our ML model achieved a sensitivity of 0.759, a specificity of 0.728, and an AUC of 0.78 using the ten variables with the highest feature impact score (FIS) to predict clonal evolution [Table 1]. All of the variables except for the presence of a PNH clone are positively correlated with sMN evolution. Nine of these variables are supported by existing literature 7,8. The feature with the third highest feature impact score, the time elapsed (days) between diagnosis and IST treatment, has not been established by previous studies and may be an important consideration in establishing optimal AA treatment guidelines. Conclusion: The strategic combination of this ML algorithm with clinical expertise has tremendous potential in improving health outcomes. Identification of patients at high risk for sMN and offering HSCT as a curative option upfront may lead to improved overall survival in AA patients.

Changes in the Immunogenetic Landscape with the Age of Disease Onset in Acquired Aplastic Anemia and Paroxysmal Nocturnal Hemoglobinuria: An Analysis of a Cohort of Adult and Pediatric Patients from the French National Reference Center for Aplastic Anemia

Over the past decade, the role of HLA molecules in the pathophysiology of immune aplastic anemia (IAA) has been highlighted by the identification of somatic clonal losses of HLA molecules on leukocytes and the enrichment of certain HLA alleles in patients with AA compared to the general population. More recently, the analysis of the HLA Evolutionary Divergence (HED) score in a large US adult cohort showed that IAA patients were carrying class II HLA molecules whose antigen-binding sites were characterized by a high degree of structural homology and that low class II HED score was associated with a higher risk of malignant progression and poorer survival (Pagliuca et al., Blood, 2021). To evaluate these findings in an independent cohort, we conducted a retrospective study of a large cohort of adult and pediatric patients diagnosed with IAA and/or PNH included in 2 different centers in Paris from the French National Center for Aplastic Anemia (RIME observatory). Patients' 2-field HLA genotyping for HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 loci and HLA-DQA1 genotype imputations were compared with those of a control cohort of 593 allogeneic donors in the context of hematologic malignancies. The MidasHLA R package was used to assess HLA-disease associations using logistic regression analysis with a dominant inheritance model (Benjamini-Hochberg adjusted p-values). HED metrics, used as a surrogate for the width of the immunopeptidome presented by the patient's HLA molecules (Pierini and Lenz, Mol. Biol. Evol., 2018) were calculated for the different HLA loci and for the entire HLA class I and HLA class II genes (mean of HED scores for the corresponding loci). The cohort consisted of 449 patients who were children (<15 years of age, n=130), adolescents and young adults (15-25 years of age, n= 141), and adults (> 25 years of age, n= 178) at the time of diagnosis. Fifty-five percent had IAA, 28% had AA with a PNH clone, 8% had hepatitis-associated AA and 9% had hemolytic PNH. The distribution of diagnoses differed across age groups, with a decreasing incidence of hepatitis-associated AA and an increasing incidence of both AA/PNH and PNH with age. We first confirmed a strong association between patients and the DRB1*15:01, DQB1*06:02 and DQA1*01:02 alleles (these 3 alleles being in strong linkage disequilibrium) in addition to a few additional alleles at the DPB1 and B loci (Figure 1). This finding was consistent with several published studies in populations of different ethnicities. Using stepwise conditional testing, we then showed that only DQB1*06:02 (and DPB1*01:01 to a much smaller extent) remained associated with the disease (OR=2.49, adjusted p-value = 7.55E-09). Interestingly, the DQB1*06:02 association was lost when analyzing the subset of pediatric patients. Among patients, we then demonstrated a strong correlation between the age at disease onset and the presence of the DQB1*06:02 (Figure 2, exact Fisher's test, p=0.0005), DQA1*01:02 (p= 0.006) and DRB1*15:01 alleles (p< 0.0001). Intriguingly, we additionally found an overrepresentation of the ancestral A*01:01 B*08:01 DRB1*03:01 haplotype among patients with primary PNH (14.6%) compared to both patients with AA/PNH (2.4%, p=0.009) and controls (6.3%, p=0.083). Next, we investigated the HED metrics at the different HLA loci. There was no significant decrease in HED scores in patients compared to control, either at the DQB1, DQA1 and DRB1 loci or for the overall HED Class II score (mean value of HED-DRB1, -DQB1 and -DPB1), even in the subsets of DRB1*15:01 or DQB1*06:02 non-carriers. This result differed from the aforementioned US study, likely due to differences in geographic inclusion areas between the 2 cohorts. To rule out bias due to the inclusion of pediatric patients in our cohort, we then focused on the adult-onset subgroup and observed similar results. However, considering the entire cohort and despite no differences between patients and controls, we identified a strong association between age at onset and HED-B and HED-Class I (mean value of HED-A, -B and -C), with the divergence increasing with age (Kendall correlation test, p=0.001 and p=0.007, respectively). Taken together, these results highlight different immunogenetic landscapes according to the age of disease onset. We can hypothesize that the immune-mediated pathophysiological mechanisms may differ with age with a preponderant role of DQB1*06:02 appearing at later age of disease onset.

Bone Marrow Failure and Inborn Errors of Immunity: An Immunogenomic Crossroad

Autoimmunity and immunodeficiency can coexist in a paradox fashion. Inborn errors of immunity (IEI) are a heterogeneous group of inherited disorders in which the immune system is disturbed, not only predisposing to infection, but also triggering autoimmunity. While investigating the presence of germline variants implicated in IEI in patients with T-LGL leukemia (Abstract #182344), we serendipitously found that these congenital defects were indeed more frequently encountered in cases with immune cytopenias and, specifically, with BMF. This finding motivated us to expand our investigation towards AA and PNH. With the advent of NGS, an increasing number of adult-onset IEI has been identified. As compared to classic IEI, autosomal dominant (AD) traits, with non-canonical, less-deleterious-effect variants, and mono-allelic defects for recessive (AR) diseases may occur in these cases, leading to incomplete penetrance and delayed, subclinical or even atypical expressivity. Few reports have described AA as an unusual manifestation of IEI and, more recently, complement factor H variants have been linked to PNH. 1 Here we hypothesized that rare germline variants predisposing to IEI may contribute to the pathogenesis of AA and its evolution to PNH. To comprehensively study the presence of IEI traits in such setting, we identified 398 AA/PNH patients, in 57 of whom we performed WGS. We assessed the presence of rare germline variants in rationally selected 120 IEI genes, spanning different immune defects. A stringent variant calling and categorization algorithm to avoid false positives was devised. We excluded variants defined by frequency >1% across genome resources. For the purpose of this study, we selected all pathogenic/likely pathogenic (P/LP) and variants of uncertain significance (VUS) overrepresented in our cohort (significant p value for Obs. vs. Exp. frequencies after multiple-testing correction). We identified 100 rare variants, 60 of which, involving 36 genes and carried by 37 patients, were significantly overrepresented. The combined allelic burden for these variants was 53% ( vs the expected 3% in normal genomes). All variants were heterozygous (median mutations/patient: 1, IQR: 1-2), and 23 and 37 defects involved genes linked to AD and AR traits, respectively. Overall, 11/60 variants, carried by 10/57 patients (18%), were classified as P/LP, and 10 were in 6 genes linked to AD traits, constituting sufficient basis for immune imbalance leading to disease. According to the immune pathway, 5 variants involved 3 IEI genes previously associated with BMF ( TERT, TINF2, DKC1), but most of them clustered in alternative IEI categories marked by T- or B-cell defects, autoimmunity, and innate or complement system defects ( FigA). A total of 4/6 genes with at least one P/LP were recurrently mutated ( TERT, AIRE, PRF1, BLK) including loci not previously related to BMF. Remarkably, 3 patients had alterations in a mutational hotspot in the gene AIRE linked to autoimmunity through dominant-negative effects. 2 We also studied genotype-phenotype correlations according to: 1) pathogenicity/inheritance pattern, and 2) immune pathway. Carriers of P/LP and AD defects (since VUS in AD vs AR genes are more likely to be of clinical relevance) had a higher proportion of autoimmunity (35 vs. 8% in non-carriers, p= .02) ( FigB). Despite there were no differences in age of diagnosis, severity of cytopenias or PNH clone presence, suboptimal response to immunosuppression was more frequently registered in carriers (63 vs. 37% in non-carriers, p= .04). Of note, variants associated with innate/complement system were more frequent in PNH (28 vs. 6% in AA, p= .04). Moreover, in patients with initial AA diagnosis, adjusting by sex, age, and PNH clone, innate/complement variants were associated with a higher risk of PNH evolution (aOR: 24, p= .02). Our study suggests that: 1) variants predisposing to immunodeficiency are overrepresented in BMF; 2) cryptic IEI traits may result in aberrant/incomplete response to otherwise easily cleared antigens, so that immune BMF may eventually arise in these cases from failed compensatory feedback mechanisms; and 3) different pathways are asymmetrically distributed between AA and PNH. We provide new insights on the immunogenomic crossroads between immunodeficiency and autoimmunity, and open the horizon of the IEI spectrum towards the fields of BMF and clonal hematological disorders.

Avatrombopag in Combination with Immunosuppressive Therapy for Hepatitis-Associated Aplastic Anemia

Hepatitis-associated aplastic anemia (HAAA) is an uncommon but distinct variant of aplastic anemia (AA), in which an episode of seronegative acute hepatitis precedes the onset of pancytopenia. Previous studies indicated that about only 20% of HAAA patients can get their hematologic partially restored after 3 months treatment with standard immunosuppressive therapy (IST). Recent studies have shown that adding thrombopoietin receptor agonist (TPO-RA) eltrombopag to standard IST can lead to a more, faster, and better hematologic responses. Nevertheless, eltrombopag is often associated with hepatic adverse effects, i.e., elevated transaminases and bilirubin, which maybe limit its use in patients with HAAA who have just experienced severe liver damage. Avatrombopag is also a TPO-RA, which was approved for the treatment of thrombocytopenia in patients with chronic liver disease (CLD) and patients with chronic immune thrombocytopenia (ITP) by the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA). The pre-clinical studies indicated that avatrombopag can promote the hematopoietic and megakaryocyte progenitor cells proliferation and differentiation by the dose-dependence. These in vitro effects are similar to those of other TPO-RAs, including Eltrombopag. Theoretically, on the basis of adequate IST, avatrombopag can also promote hematopoietic recovery in patients with aplastic anemia just as Eltrombopag works. If this is true, then avatrombopag should be particularly suitable for HAAA due to its favorable hepatic adverse effect profile. Nevertheless, avatrombopag added to standard IST as first-line treatment for aplastic anemia could increase the rapidity and strength of hematologic response is still unknown. Herein, we retrospectively reviewed 8 consecutive cases of patients with hepatitis-associated aplastic anemia who were treated with avatrombopag plus IST regimen. From September 2021 to December 2022, eight patients with hepatitis-associated aplastic anemia treated with avatrombopag in combination with standard IST were enrolled in this study. Among them, seven patients were diagnosed as VSAA and one SAA. The median age was 17 (9-20) years old and the median absolute neutrophil count was 0.09 (0.02-0.39) ×10 9/L. The median time from onset of hepatitis to hematopoietic failure was 2 (0.5-5.5) months, and at the time of beginning of IST all the eight patients had their liver biochemical testing returns to normal. The karyocytes were normal in all patients. One patient presented positive PNH clone at the diagnosis of HAAA, with PNH clone size of 4.7%. Avatrombopag was incorporated into day 1 of standard immunosuppressive therapy with p-ATG and cyclosporine for all the eight HAAA patients. The dose of avatrombopag was 40 mg/d at least 6 months. Dosage adjusting of avatrombopag was according to patient's platelet counts and adverse events. Encouragingly, four of the eight patients (50.0%) achieved hematologic response (HR) with two (20.0%) getting complete response (CR) at 3 months after IST and avatrombopag. Between 3 and 6 months after IST, the other two patient entered their hematologic responded. Then, six of eight (75.0%) patients got HR at 6 months, including 3 CR, 2 good partial hematologic response (GPR), and 1 partial response (PR). Unfortunately, one patient who was diagnosed as very severe HAAA, was without getting hematologic response and died at 118 days after treatment due to gastrointestinal infection. We evaluate the side effects of avatrombopag and graded according to the Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. No hepatic adverse events were observed in all the eight patients, that indicated avatrombopag may not increase the hepatic burden and is safe for HAAA. The median follow-up time was 371(118-665) days. Except for one patient who died within 6 months, six cases achieved hematologic response, with 4 CR and 2 GPR. There is no clonal evolution, i.e., MDS, AML, and PNH, was observed. In conclusion, avatrombopag may be used as a safe and effective TPO-RA, added to IST for the treatment of hepatitis-associated aplastic anemia, which maybe improve the efficacy and the strength of hematologic response in HAAA.

Allogeneic Hematopoietic Stem Cell Transplantation for Classical Paroxysmal Nocturnal Hemoglobinuria

Classical paroxysmal nocturnal hemoglobinuria (cPNH) is a rare acquired clonal hematopoietic cell disease. Eculizumab is effective in PNH patients, though, it must be administered indefinitely. As the technology of allogeneic hematopoietic stem cell transplantation (allo-HSCT) improves in recent years, what is the position of HSCT in treatment of cPNH? This study discusses the efficacy and safety of allo-HSCT in cPNH. The clinical data of 5 cPNH patients who underwent allo-HSCT from 2019 to 2022 were collected. All patients were diagnosed cPNH according to guidelines from International PNH Interest Group. Indications for HSCT were transfusion-dependent and nonavailability of eculizumab. The cohort was all male, with a median age of 26 years (range, 26 to 46 years) at the time of transplantation. All patients had documented hemolysis and no thrombosis during monitoring. The median time from diagnosis to allo-HSCT was 5.5 years (range, 3.6 to 18 years). The median PNH granulocyte clone size was 96.3% (range, 90% to 99.7%), and the median lactate dehydrogenase (LDH) level was 2224IU/L (8.896-fold of the upper limit of normal). All patients were detected bone marrow hyperplasia by trephine biopsy. The stem cell source came from 4 haploidentical donors and 1 HLA-identical sibling donor. Among the 5 patients, 3 patients underwent myeloablative conditioning (MAC) and 2 patients underwent reduced-intensity conditioning (RIC) treatment. None of the patients experienced primary implantation failure. Neutrophil implantation time was 15 (13-21) days, and platelet implantation time was 24 (13-60) days. 3 patients developed grade II acute graft-versus-host disease (aGVHD). No patients developed grade Ⅲ-Ⅳ aGVHD. 2 patients developed localized chronic graft-versus-host disease (cGVHD) involving lung and skin respectively, and no patients developed extensive cGVHD. PNH clones turned negative in all patients after 2 months (range, 1 to 3 months) of transplantation. At a median follow-up of 16 (6-34) months, 1 patient died of relapse and infection. The remaining 4 patients survived, of which 2 patients had discontinued all drugs. cPNH has its own unique clinical features. Allo-HSCT could cure cPNH patients who can withdrawal from treatment of all medication. Haploidentical donor HSCT is an acceptable choice for cPNH, and RIC constitutes a valid option.

Bone Marrow Versus Peripheral Blood in HLA-Matched Sibling Transplantation: Does the Stem Cell Source Matter in Severe Aplastic Anemia?

Background Selecting a graft source is critical in HLA-matched sibling donor hematopoietic stem cell transplantation (MSD-HCT) for severe aplastic anemia (SAA). Since graft-versus-host-disease (GVHD) is the major cause of mortality after MSD-HCT, and SAA patients do not need the graft-versus-leukemia effect, the selection of a suitable source of stem cells becomes critical. Although bone marrow (BM) is the preferable stem cell source based on the registry-based studies, mobilized peripheral blood stem cells (PBSC) may offer faster engraftment with rapid immune reconstitution. Moreover, the recent advances in conditioning regimens have significantly reduced transplantation-related complications. Here, we compared the long-term outcomes of BM vs PBSC as a graft source in adult patients with SAA who received MSD-HCT with the reduced intensity conditioning (RIC) regimen. Method We conducted a retrospective analysis of 171 consecutive SAA adult patients who received MSD-HCT with Flu6/Cy2/ATG4 RIC regimen (Flu, 30 mg/m 2 IV for 6 days: Cy, 50 mg/kg IV for 2 days; rabbit ATG, 2.5 mg/kg IV for 4 days) or total nodal irradiation (TNI) of 750 cGy combined with ATG as GVHD prophylaxis (TNI-750/ATG) from March 2002 to March 2021 at Seoul St. Mary's Hospital, Seoul, Korea. BM was the primary graft source, but PBSC was used in donors who refused BM harvest, were unsuitable for general anesthesia, or received TNI-750/ATG conditioning. The primary endpoint of this study was to compare the overall survival (OS), and the secondary endpoints included speed of hematologic recovery, primary or secondary graft failure (GF), and failure-free survival (FFS) between the BM and PBSC group. Results The median patient age in the BM group was 38 years (range, 15 - 59) and 44 years (range, 19 - 64) in the PBSC group (p=0.005). There were no differences of baseline characteristics between the two groups except for a donor-to-recipient sex discrepancy (female to male, BM vs. PBSC; 16.4% vs. 30.9%, p=0.029). The majority of BM group (114/116, 98.2%) received the Flu6/Cy2/ATG4 RIC regimen, while 38.2% (21/55) of PBSC group received TNI-750/ATG for the conditioning (p<0.001). The PBSC contained a median 4.71 (range, 1.22 - 17.03) x 10 6/kg CD34 + cells and 368.82 (range, 25.12 - 1234.70) x 10 6/kg CD3 + cells which were about 1.7-fold (BM 2.80 x 10 6/kg CD34 + cells; range, 0.25 - 10.05) and 10-fold (BM 39.61 x 10 6/kg CD3 + cells; range, 1.34 - 463.68) higher than the BM graft (p<0.001). Consequently, the PBSC group exhibited faster engraftment of neutrophils (median 13 vs. 11 days, p<0.001) and platelets (median 18 vs. 12 days, p=0.012). However, there were no differences in the incidence of secondary GF (19.0% vs. 12.7%, p=0.386) and donor-type aplasia (4.3% vs. 10.9%, p=0.178) between two groups. Furthermore, the incidence of acute grade I-II (8.6% vs. 10.9%, p=0.619) and moderate to severe chronic graft-versus-host disease (GVHD) (3.5% vs. 5.5%, p=0.542) were also not significantly different between two groups. However, the BM group has shown more CMV reactivation (44.0% vs. 24.9%, p=0.005), and higher incidence of respiratory infection (14.7% vs. 3.6%, p=0.037) than those of the PBSC group. The incidences of transplantation-related complications between the BM and PBSC group are summarized in Table. The 10-year OS (93.8% vs. 98.1%, p=0.246) and FFS (65.7% vs. 69.0%, p=0.455) were comparable (Figure). Moreover, there was no significant difference in FFS between BM and PBSC group by potential risk factors of patient and donor age at MSD-HCT, sex incompatibility, ABC mismatch, PNH clone presence, HCT-CI ≥3, infused CD34 + cell count, interval from diagnosis to transplant, prior IST history, heavy transfusion before allo-HCT, or severity of AA. Conclusions Our results suggest that survival outcomes of MSD-HCT showed no difference between BM and PBSC as graft sources, and using PBSC may be an efficient alternative offering faster engraftment without significant difference in graft failure or transplantation-related adverse events such as GVHD, and survival. Current analysis and results were drawn from the Asian population with a fludarabine-based conditioning regimen and therefore, need to validate in the Western population. In addition, well-designed prospective studies in a larger cohort are required to confirm our findings.

Deciphering the Differential Diagnosis of Inherited Versus Acquired Bone Marrow Failure Syndromes: A Large Cohort Study

The wide overlaps in clinical and hematologic features of inherited (iBMF) and acquired (aBMF) bone marrow failures are often the cause of diagnostic difficulties and delay in proper therapeutic approaches. Screening tests for iBMF are complex and expensive and have limited availability. Data-driven machine learning algorithms may reveal useful tools in clinical practice to guide differential diagnosis. We collected data about clinical, laboratory and natural history of 597 patients with BMF (55 iBMF; 542 aBMF). iBMFs were typically younger (30 y, 0.1-71 vs 44 y, 2-85, p <0.001) and presented higher baseline blood counts (reticulocytes 62x10^9/L, 31-205 vs 35x10^9/L, 0-216; neutrophiles 1.4x10^9/L, 0-4.3 vs 0.6x10^9/L, 0-4.6; platelets 93x10^9/L, 7-356 vs 23x10^9/L, 1-355, p <0.001), and more cytogenetic aberrations (32% vs 13%, p 0.002). Conversely, PNH clones were detected and confirmed overtime mainly in aBMF (51% vs 9% p <0.001), with median clone size of 0.6% (range 0.02-3.8) in iBMF. By NGS analysis, no differences in acquired myeloid gene mutations incidence were detected between the groups (25% vs 13%, p 0.1). Evolution to MDS occurred in 20% of iBMF vs 11% of aBMF (p 0.04), and to AML in 3.6% vs 1.5% (p 0.2). Time between diagnosis and evolution was shorter in iBMF (2 y, 0-14 vs 5.4 y, 0.2-32 p 0.01). Survival was similar between the 2 groups with median values of 7 y (95%CI, 6.1-7.6), reaching 8 y (95%CI, 6.9-9.2) in transplanted patients. Factors significantly associated with a better survival were female gender (HR: 0.73, 95%CI: 0.59-0.90, p 0.004), BMT (HR: 0.62, 95%CI: 0.47-0.83, p 0.001), number of therapy lines (HR: 0.67, 95%CI: 0.51-0.87, p 0.003), response to 1 st line (HR: 0.53, 95%CI: 0.41-0.69, p < 0.001), and evolution to PNH (HR: 0.61, 95%CI: 0.40-0.96, p 0.03). Conversely, a lower survival was associated with older age: patients aged 31-60 had 2-fold risk of death compared to those under 30 (HR 1.8, 95%CI 1.15-2.75, p <0.001); patients over 60 had a 3.2 times higher risk compared to under 30 (HR 3.2, 95%CI 2.35-4.45, p <0.001). Presence of trilineage cytopenia also predicted poorer survival (HR: 2.92, 95%CI: 2.25-3.79, p <0.001 versus single- or bi-lineage cytopenias). No clear association with survival was found regarding karyotype and presence of mutations at myeloid NGS panel (Table 1). Finally, via a 10-folds cross-validation, we built a multivariate logistic model to predict the probability of having an iBMF. A dataset composed of 139 patients (120 aBMF and 19 iBMF) was used to train and validate the model, while subjects with missing information were excluded from the analysis. The obtained classifier was tested on an independent test set, resulting in 96% (81-100) accuracy, with 26/27 patients correctly assigned to their true class, 100% sensitivity and 96% specificity and having area under the ROC curve (AUC) equal to 0.97. Factors showing an impact on the predictive model were as follows: higher Hb levels (OR 2.35, range 0.98-5.60, p 0.05), reticulocytes (OR 1.01, range 0.99-1.04, p 0.29), platelets (OR 1.05, range 1.01-1.09, p 0.005), and erythropoietin (OR 1.0, range 1.00-1.01, p 0.009) were associated with IBMF; whilst older age (OR 0.91, range 0.84-0.99, p 0.03), normal NGS myeloid panel (OR 0.09, range 0.01-1.52, p 0.09), and the presence of a PNH clone (OR 0.46, range 0.04-4.83, p 0.52) showed a negative association (Figure 1). In conclusion, iBMF and aBMF differ in clinical presentation and risk of clonal evolution, and deserve specific treatments (IST in aBMF vs BMT and androgens in iBMF) and special considerations (i.e. choice of BMT donor and conditioning in iBMF). The proposed model includes few and routinely available variables and may be a practical tool to be implemented in the clinic, though it will require validation on an independent cohort to confirm its feasibility and prediction accuracy.

Haploidentical Hematopoietic Cell Transplant with Post-Transplant Cyclophosphamide Expedites Donor-Derived Immune Reconstitution in a Pediatric Severe Aplastic Anemia Cohort

Severe aplastic anemia (SAA) is a life-threatening, idiopathic process involving the immune-mediated destruction of hematopoietic stem cells. Treatment involves immunosuppressive therapy (IST) and/or allogeneic hematopoietic cell transplantation (HCT). The latter has recently been optimized with use of a post-transplant cyclophosphamide (PTCy) based graft-versus-host disease (GvHD) platform, permitting successful HCT with only half-HLA-matched haploidentical donors. In pediatrics, this platform results in >90% overall survival with low rates of GvHD and graft failure. However, reports on donor chimerism and immune reconstitution are limited. We hypothesized that a PTCy platform would expedite CD3 donor engraftment and CD4 T cell recovery. In a phase III clinical trial, we treated three cohorts of pediatric (age </=25 years) patients with SAA requiring alloHCT: MUD, MSD, and HAPLO. The MUD cohort included n=15 recipients of 7-8/8 HLA-matched unrelated donor grafts (n=5 7/8, n=10 8/8) with median age 14 years (range 2-17 years), following cyclophosphamide (Cy) 50 mg/kg, fludarabine (Flu) 200 mg/m 2, rabbit anti-thymocyte globulin (rATG) 9 mg/kg total dose rabbit and low dose total body irradiation (TBI) at 200 cGy. The MSD cohort included n=9 recipients of 8/8 HLA-matched sibling donor grafts with median age 15 years (range 12-24 years), following Cy 200 mg/kg and 9 mg/kg rATG. The HAPLO cohort included n=4 recipients of 4/8 HLA-matched related donor grafts with median age 8.5 years (range 2-21 years) following Cy 29 mg/kg, Flu 150 mg/m 2, 9 mg/kg rATG, and low dose TBI at 200 cGy. GvHD prophylaxis consisted of 30 days of mycophenolate mofetil, and 100 days of a calcineurin inhibitor (followed by an 8-10 week taper), with the HAPLO cohort additionally receiving post-transplant Cy (PTCy) 50 mg/kg/day on days +3 and +4. Peripheral blood lymphocyte counts and peripheral blood and bone marrow donor chimerism were evaluated on days +100, +180 and +365. Recipient characteristics were largely equivalent between cohorts, with 43% receiving IST prior to HCT. Clonality was overrepresented in the HAPLO cohort (2 of 4, 1 with monosomy 7 myelodysplastic syndrome and 1 with a large PNH clone), with only 2 of 15 in the MUD and none in the MSD cohorts. Active controlled infections at the time of HCT were additionally overrepresented in the HAPLO cohort, with disseminated pseudomonas in 1. The MUD cohort had 1 with Rhizopus invasive sinusitis (none in the MSD cohort). Achievement of neutrophil engraftment was statistically significantly delayed in the HAPLO cohort at a median of 17 days compared to 12 for MSD and 14 for MUD, p=0.004), however there were no episodes of primary or secondary graft failure. With only a single late death from intracranial hemorrhage in the MUD cohort, OS was equivalent at 2 years (100% in MSD and HAPLO cohorts, 93.3% in MUD). GvHD rates were low with only 1 (MUD) experiencing grade II acute GvHD, and 1 (MUD) experiencing mild chronic GvHD. Immune reconstitution varied by cohort with statistically significantly greater CD3 peripheral blood donor chimerism (p<0.001) as well as CD4 (p=0.04) and CD8 (p=0.03) T cell counts over time for the HAPLO recipients (CD3 chimerism in Fig 1, CD4 count in Fig 2; 2-way ANOVA fixed effects model). Considering a CD4 >200 cell/uL as clinically meaningful, we assessed the proportion of each cohort achieving this milestone at each timepoint. While each cohort had an increase in proportion achieving CD4>200 over time, the HAPLO cohort reached 50% with CD4 >200 at day +100, compared to 11% MUD and 0% MSD (p=0.09). At day +180, the proportion with CD4 >200 were 36% MUD, 25% MSD and 75% HAPLO (1 yr: 90% MUD, 100% MSD and HAPLO). Donor availability remains a challenge to successful transplant for patients with SAA, and haploidentical matches overcome donor limited accessibility. PTCy as GvHD prophylaxis is associated with low incidences of graft failure and severe GvHD. While PTCy might be anticipated to inhibit donor T cells and immune reconstitution, our data, albeit limited by small cohorts, demonstrates superior and more efficient CD3 donor engraftment and CD4 T cell count recovery following HAPLO HCT with PTCy compared to non-PTCy based MUD and MSD cohorts.

Deciphering treatment patterns in non-severe/moderate aplastic anemia: an international observational study

Non-severe aplastic anemia is a rare bone marrow failure disorder characterized by variable degrees and combination of cytopenias, with limited data on management and outcome. We describe a large multicentric series of 259 patients, focusing on clinical and molecular features, treatment, evolution, and survival. The majority required treatment with cyclosporine (CyA) alone (N = 84) or in combination with anti-thymocyte globulin (ATG,44) or eltrombopag (20), eltrombopag alone (10), or others (25) including androgens. Similar outcomes were observed across different strategies, with a 6-month overall response rate of 73% for CyA, 74% for ATG plus CyA, 68% for CyA plus eltrombopag, 87% for eltrombopag, and 79% for others. Notably, 56 patients (39%), mainly receiving CyA plus eltrombopag, achieved a trilineage response (p = 0.02). Progression to myeloid neoplasms was limited (8%) and not related to mutational status. Hemolytic PNH developed in 10% of cases, being predicted by detection of small clones at diagnosis. Survival was negatively impacted by age, male gender, LDH, platelets/erythrocyte transfusion need, and somatic mutations by NGS, and positively by higher neutrophils at diagnosis, PNH clones, and trilineage response at 6 and 12 months. Multivariable analysis confirmed the detrimental role of age and the favorable association with PNH clone and trilineage response at 6 months.

Paroxysmal nocturnal haemoglobinuria presenting as cortical cerebral venous thrombosis

Abstract Paroxysmal nocturnal haemoglobinuria (PNH) arises as a consequence of mutation of phosphatidylinositol N-acetylglucosaminyltransferase subunit A gene that is required for synthesis of the glycosylphosphatidylinositol proteins to the cell surface of blood cells. Patients with PNH usually present with features of chronic unexplained anaemia, but can also present with bone marrow failure and thrombosis at unusual sites. We present the case of a 27-year-old who presented with complaints of headache and easy fatigability, on evaluation was found to have coombs negative haemolytic anaemia and brain magnetic resonance venogram revealed thrombosis in the left transverse and sigmoid sinuses. On further investigation, found to have PNH clone in both red and white blood cell lineages and diagnosed as PNH. Hence, any patient presenting with chronic non-immune intravascular haemolysis, unexplained anaemia and unusual thrombosis should alert the physician to consider PNH as a differential diagnosis to avoid delay in diagnosis and better management.

Swiss Survey on current practices and opinions on clinical constellations triggering the search for PNH clones.

This study aimed to investigate clinical indications prompting PNH clones' assessment and physician's accessibility of a flow cytometry facility, and also to understand clinical attitudes on the follow-up (FU) of patients with PNH clones. The survey includes 16 multiple-choice questions related to PNH and targets physicians with a definite level of experience in the topic using two screener questions. Opinion on clinical management was collected using hypothetical clinical situations. Each participant had the option of being contacted to further discuss the survey results. This was an online survey, and 264 physicians were contacted through email once a week for 5 weeks from September 2020. In total, 64 physicians (24.2%) from 23 institutions participated (81.3% hematologists and 67.2% from university hospitals). All had access to flow cytometry for PNH clone testing, with 76.6% having access within their own institution. The main reasons to assess for PNH clones were unexplained thrombosis and/or hemolysis, and/or aplastic anemia (AA). Patients in FU for PNH clones were more likely to be aplastic anemia (AA) and symptomatic PNH. In total, 61% of the participants investigated PNH clones repetitively during FU in AA/myelodysplastic syndromes patients, even when there was no PNH clone found at diagnosis, and 75% of the participants tested at least once a year during FU. Opinions related to clinical management were scattered. The need to adhere to guidelines for the assessment, interpretation, and reporting of PNH clones emerges as the most important finding, as well as consensus for the management of less well-defined clinical situations. Even though there are several international guidelines, clear information addressing specific topics such as the type of anticoagulant to use and its duration, as well as the indication for treatment with complement inhibitors in some borderline situations are needed. The analysis and the discussion of this survey provide the basis for understanding the unmet needs of PNH clone assessment and clinical practice in Switzerland.